A Dual-Signaling Electrochemical Aptasensor Based on an In-Plane Gold Nanoparticles–Black Phosphorus Heterostructure for the Sensitive Detection of Patulin

Patulin (PAT), a type of mycotoxin existing in foodstuffs, is harmful to food safety and human health. Thus, it is necessary to develop sensitive, selective and reliable analytical methods for PAT detection. In this study, a sensitive aptasensor based on a dual-signaling strategy was fabricated, in which a methylene-blue-labeled aptamer and ferrocene monocarboxylic acid in the electrolyte acted as a dual signal, for monitoring PAT. To improve the sensitivity of the aptasensor, an in-plane gold nanoparticles–black phosphorus heterostructure (AuNPs-BPNS) was synthesized for signal amplification. Due to the combination of AuNPs-BPNS nanocomposites and the dual-signaling strategy, the proposed aptasensor has a good analytical performance for PAT detection with the broad linear range of 0.1 nM–100.0 μM and the low detection limit of 0.043 nM. Moreover, the aptasensor was successfully employed for real sample detection, such as apple, pear and tomato. It is expected that BPNS-based nanomaterials hold great promise for developing novel aptasensors and may provide a sensing platform for food safety monitoring.


Introduction
Patulin (a polyketide lactone 4-hydroxy-4H-furo(3,2c) pyran-2(6H)-one, PAT) belongs to a class of toxic compounds known as mycotoxins and is a common contaminant in food and feed. It is a toxic metabolite of at least 60 different species of fungi, such as Penicillium expansum (P. leucopus), P. patulum (P. urticae, P. griseofulvum), P. crustosum and A. clavatus [1]. Therefore, humans are likely to be exposed to such toxic substances, which may cause various toxic effects, such as lung and liver bleeding, kidney damage, neurotoxicity, teratogenicity and possible carcinogenicity [2]. Accordingly, the levels of PAT in food are controlled by a set of regulations. Marín et al. [2] reported that the maximum level of patulin in pears and apples is 126 mg/kg in the Spanish market, which is twice as high as the European Union (EU) standard. The World Health Organization and the Food and Agriculture Organization recommend a maximum daily level of PAT of 0.4 µg/kg body weight/day [3]. In China, patulin is also a common contaminant in a variety of fruits and products. The rate of patulin detection was 76.9% in 401 fruit products from eight provinces in China [4]. Hence, different analytical techniques have been explored for highly sensitive and accurate detection of PAT, such as chromatography [5][6][7][8], colorimetry [9] and immunoassays [10,11]. However, these methods required heavy instrumentation and complex technology, especially concerning the instability of antibodies in immunoassays [12]. Electrochemical sensors are a powerful technique because of their fast response time, low cost and excellent flexibility.
At present, aptamers, a type of DNA or RNA, can recognize various target molecules (metal ions, small molecules, proteins, etc.) with high affinity and have drawn interest in sensor design [13,14]. Combining aptamers and electrochemical methods, electrochemical aptasensors possess an excellent performance. However, most electrochemical aptasensors have only one signal readout: either a "signal off" or "signal on". The sensitivity of onesignal electrochemical sensors is inevitably limited, owing to signal fluctuation [15,16]. A dual-signaling strategy can solve these drawbacks. Numerous studies of electrochemical aptasensors with dual-signal channels have been reported to realize the high-performance detection of proteins and enzymes [17,18]. In dual-signaling electrochemical sensors, two electrochemical signals are dependent on targets, and their peak intensities show associated changes simultaneously. Therefore, dual-signaling electrochemical sensors exhibit a promising performance, with benefits such as a lower limit of detection and higher sensitivity.
To improve the sensitivity of electrochemical sensors, many nanomaterials have been applied for the amplification of signal. Among the various nanomaterials, two-dimensional (2D) nanomaterials have attracted widespread attention due to their unique physicochemical properties [19]. The first 2D nanomaterials appeared in 2004, when graphene was exfoliated from graphite by the mechanical cleavage technique [20,21]. Black phosphorus (BP), an allotrope of phosphorus with an orthorhombic lattice, is emerging as an important successor to 2D nanomaterials [22]. BP nanosheets (BPNS) can be exfoliated from bulklayered crystal by breaking down its relatively weak van der Waals interactions, where each phosphorus atom forms covalent bonds with three neighboring atoms, forming a puckered orthorhombic structure [23,24]. The intriguing physical properties such as the high chargecarrier mobility, large on/off ratio, significant anisotropy and layer-dependent band gap have spurred tremendous interest in BP [25][26][27][28]. Owing to a direct, tunable band gap and no cytotoxicity, BPNS was used to explore photocatalyst and biomedical applications and more [29][30][31]. In order to improve the properties of BPNS and broaden their application, some nanocomposites based on BPNS have been synthesized by hybridization, doping or functionalization [32]. Sun et al. developed BPNS/graphene for sodium-ion batteries, which was self-assembled by intercalating BPNS between graphene layers via van der Waals interactions [33]. Hersam and co-workers reported that BPNS was functionalized by aryl diazonium through the formation of P-C covalent bonds and applied for field effect transistors [34]. Currently, the application of BPNS or BPNS-based nanocomposites in electrochemical sensors has not been adequately explored, which may be because of the instability of BP. Our group prepared gold nanoparticles-BPNS by electrostatic attraction for PAT electrochemical sensing [35]. However, this preparation method requires a more intensive experimental process and cannot enhance the stability of BP. The preparation of monodispersed BP nanosheets depends on long-time ultrasonic treatment or shear exfoliation, during which the BP layers are gradually cleaved and defects are generated on the edges and surface. These defects not only lead to the rapid degradation of the BP nanosheets, but also undermine the conductivity and electrochemical activity, thus causing problems in many applications-especially long-term catalysis. It is a good strategy to design the heterostructures to tailor the properties of 2D nanomaterials [36]. Due to the intrinsic advantages of the in-plane heterostructures, the in-plane charge carrier mobility of 2D materials is much higher than that between interlayers.
In this work, an in-plane gold nanoparticles-BPNS (AuNPs-BPNS) heterostructure was synthesized, in which the high chemical activity of the defect sites in BPNS can edgeselectively reduce HAuCl 4 , and the formed AuNPs not only occupy the defect sites but also provide effective immobilization sites for the aptamer. Then, the obtained AuNPs-BPNS nanocomposites were used to construct a simple dual-signaling electrochemical aptasensor for sensitive and accurate detection of PAT (Scheme 1). The dual-signal was obtained from a methylene blue (MB)-labeled aptamer and ferrocene monocarboxylic acid (FMCA) in the electrolyte. The MB-labeled PAT aptamer self-assembled on the surface of pre-prepared AuNPs-BPNS/glassy carbon electrode (GCE) through the Au-S bond. Then, single-stranded DNA complementary to the aptamer formed a rigid double strand. In the presence of PAT, the aptamer recognized PAT and the complementary Foods 2023, 12, 846 3 of 14 DNA was released. As a result of the conformational change in the aptamer, the MB tag came close to the surface of the electrode and the diffusion of FMCA was hindered due to the increased steric resistance. Thus, the peak current of MB increased and FMCA decreased, producing "signal on" and "signal off". In this sensing system, because of the electronic property of BPNS and the sensitivity of the dual-signaling strategy, the proposed aptasensor exhibited an excellent performance for PAT detection. Moreover, the prepared aptasensor could also be applied in real sample detection. The developed sensing platform exhibited a great potential for the sensitive detection of other aptamerspecific binding contaminants of foodstuffs.
obtained from a methylene blue (MB)-labeled aptamer and ferrocene monocarbox (FMCA) in the electrolyte. The MB-labeled PAT aptamer self-assembled on the s pre-prepared AuNPs-BPNS/glassy carbon electrode (GCE) through the Au-S bon single-stranded DNA complementary to the aptamer formed a rigid double stran presence of PAT, the aptamer recognized PAT and the complementary D released. As a result of the conformational change in the aptamer, the MB tag ca to the surface of the electrode and the diffusion of FMCA was hindered du increased steric resistance. Thus, the peak current of MB increased and FMCA de producing "signal on" and "signal off". In this sensing system, because of the e property of BPNS and the sensitivity of the dual-signaling strategy, the p aptasensor exhibited an excellent performance for PAT detection. Moreover, the p aptasensor could also be applied in real sample detection. The developed sensing exhibited a great potential for the sensitive detection of other aptamer-specific contaminants of foodstuffs. Scheme 1. Assembly diagram of the electrochemical dual-signaling aptasensor for PAT d
The PAT aptamer and DNA sequence employed in this research were pu from TaKaRa (Dalian, China). The sequences were as follows: PAT aptamer: 5′-MB-CCCGGCCCGCCAACCCGCATCATCTACACTGATATTTTACCTT SH-3′.

Apparatus and Characterization
All of the electrochemical experiments were performed with an electrochemical workstation (Guangzhou Insens Sensor Technology Co. Ltd, Gua China). The three-electrode system consisted of a modified GCE (3 mm in diam the working electrode, a platinum wire as the auxiliary electrode, and Ag/AgCl (s Scheme 1. Assembly diagram of the electrochemical dual-signaling aptasensor for PAT detection.

Apparatus and Characterization
All of the electrochemical experiments were performed with an IGS1130 electrochemical workstation (Guangzhou Insens Sensor Technology Co. Ltd, Guangzhou, China). The three-electrode system consisted of a modified GCE (3 mm in diameter) as the working electrode, a platinum wire as the auxiliary electrode, and Ag/AgCl (saturated KCl) electrode as the reference electrode; this system was used in all electrochemical investigations. Transmission electron microscopy (TEM) images were acquired with a Hitachi H-7500 high-resolution transmission electron microscope (Tokyo, Japan) using an accelerating voltage of 200 kV. UV-Vis absorption spectra were acquired on a Cary 50 Scan UV-Vis spectrophotometer (Varian, Australia). X-ray photoelectron spectra (XPS) were obtained with an ESCALAB 250 Xi XPS system from Thermo Scientific (Waltham, MA, USA). Raman spectra were recorded using a Renishaw Micro-Raman spectroscopy system (London, England) with an excitation wavelength of 514 nm.

Preparation of BPNS
For preparation of BPNS, 5 mg of BP crystal was dispersed in 50 mL saturated sodium hydroxide solution of NMP under sonication at room temperature for 12 h. Then, the solution was centrifuged at 4000 rpm for 10 min and the supernatant was collected. Finally, BPNS were obtained by centrifuging the supernatant at 12,000 rpm for 15 min and dispersed in water as a brown solution.

Preparation of AuNPs-BPNS
AuNPs were modified on BPNS by in situ reduction. Briefly, 250 µL of 25 mM HAuCl 4 was added to 1 mL of 1 mg/mL BPNS solution and stirred for 1 h in oxygen-free dark conditions. The products were separated by centrifugation and washed with deionized water. Finally, the obtained product was dispersed in water and stored at 4 • C.

Preparation of the Modified Electrodes
The GCE was polished to a mirror-like surface by 1.0 and 0.3 µm alumina slurries, respectively, and completely cleaned in ethanol and ultrapure water by ultrasonication. Then, 10 µL of a 1 mg/mL AuNPs-BPNS solution was dropped into GCE and dried in air at room temperature. Before the aptamer was fixed, the aptamer was added to phosphatebuffered saline solution (PBS; pH 7.0) containing 10 mM TCEP for treatment for 1 h to reduce the presence of disulfide bonds, and then the treated aptamer was dropped onto AuNPs-BPNS/GCE for 12 h. The electrode was then immersed in 1 mM MCH for 1 h to block the unbound sites. Finally, 10 µL of 1 µM cDNA was dropped onto the electrode surface for 1.5 h to obtain the final modified electrode cDNA/aptamer/AuNPs-BPNS/GCE.

Electrochemical Detection of PAT
For the detection of PAT, the modified electrode was immersed in 20 µL of a fixed concentration of PAT for 60 min under room temperature and then rinsed with the PBS to wash away the unbound PAT. The obtained electrode was then immersed in PBS (pH 7.4) containing 1 µM FMCA and investigated by cyclic voltammetry (CV) and cyclic voltammetry (SWV). The selective experiments for hydroxymethylfurfural (HMF), aflatoxin B1 (AFB1), aflatoxin M1 (AFM), ochratoxin A (OTA) and zearalenone (ZEN) were performed under the same conditions. All experiments were performed at room temperature and measured three times.

Pretreatment of Food Samples
In this experiment, fresh and infected apples, pears, and tomatoes acted as real food samples. The pretreatment procedures were performed according to previous work [37]. Apples, pears and tomatoes were smashed separately, then 2.5 g of the obtained sample mash was added into 10 mL H 2 O and extracted by ultrasonication for 1 h. The supernatant from each sample was transferred to a centrifuge tube and centrifuged at 6000 rpm for 5 min. Then, the extracts were collected. Finally, the supernatant and different concentrations of PAT were added to PBS.

Characterization of the AuNPs-BPNS
The morphologies of the as-prepared BPNS and AuNPs-BPNS were characterized by TEM technologies. The TEM micrograph of BPNS is presented in Figure 1A. BPNS presents a smooth surface with some wrinkles, which is a 2D nanostructure. Figure 1B shows the TEM patterns of AuNPs-BPNS nanocomposites. AuNPs were successfully synthesized on the surface of BPNS without other reduction and the inset of Figure 1B  shows an average diameter of AuNPs of about 21 nm. Elemental analyses of the AuNPs-BPNS nanocomposites entailed EDX mappings of P, Au and O, where P and O originated from BPNS and Au indicated the presence of AuNPs ( Figure 1C). Notably, AuNPs were uniformly distributed on the entire BPNS surface. As shown in Figure 1D, the EDX analysis further revealed the existence of P, Au and O in the nanocomposites.
The morphologies of the as-prepared BPNS and AuNPs-BPNS were chara by TEM technologies. The TEM micrograph of BPNS is presented in Figure 1A presents a smooth surface with some wrinkles, which is a 2D nanostructure. Fi shows the TEM patterns of AuNPs-BPNS nanocomposites. AuNPs were succ synthesized on the surface of BPNS without other reduction and the inset of Fi shows an average diameter of AuNPs of about 21 nm. Elemental analyses of the BPNS nanocomposites entailed EDX mappings of P, Au and O, where P and O or from BPNS and Au indicated the presence of AuNPs ( Figure 1C). Notably, AuN uniformly distributed on the entire BPNS surface. As shown in Figure 1D, t analysis further revealed the existence of P, Au and O in the nanocomposites. The effect of HAuCl4 concentration on the morphology of AuNPs-BPNS w investigated. As shown in Figure 2A, when 10 mM HAuCl4 was added, only amount of AuNPs were generated on the surface of the BPNS. As the concentration increased to 20 mM ( Figure 2B), the amount of AuNPs on the BPNS increased. A satisfying amount of AuNPs-BPNS could be obtained once the conce of HAuCl4 was increased to 25 mM ( Figure 2C). It can be seen that AuNPs were un deposited on the surface of BPNS without obvious aggregation, which may be the defect of BPNS is exactly occupied by the AuNPs. However, when 30 mM was added ( Figure 2D), the AuNPs showed obvious aggregation. Therefore, HAuCl4 was selected to prepare the AuNPs-BPNS nanocomposites. The effect of HAuCl 4 concentration on the morphology of AuNPs-BPNS was also investigated. As shown in Figure 2A, when 10 mM HAuCl 4 was added, only a small amount of AuNPs were generated on the surface of the BPNS. As the HAuCl 4 concentration increased to 20 mM ( Figure 2B), the amount of AuNPs on the BPNS surface increased. A satisfying amount of AuNPs-BPNS could be obtained once the concentration of HAuCl 4 was increased to 25 mM ( Figure 2C). It can be seen that AuNPs were uniformly deposited on the surface of BPNS without obvious aggregation, which may be because the defect of BPNS is exactly occupied by the AuNPs. However, when 30 mM HAuCl 4 was added ( Figure 2D), the AuNPs showed obvious aggregation. Therefore, 25 mM HAuCl 4 was selected to prepare the AuNPs-BPNS nanocomposites. UV-Vis spectroscopy was conducted to characterize the formation proces AuNPs. Figure 3A shows the UV-Vis spectrum of BPNS with different amounts o HAuCl4. As the amount of BPNS increased, the absorption peaks of the AuNPs in at 536 nm, demonstrating that the formation of the AuNPs is related to th Additionally, the UV-Vis spectrum of HAuCl4 added to BPNS with the increase in time is shown in Figure 3B. The absorption peak intensity of the AuNPs increas the increase in time, and the color of the solution changed to different shades color. The successful synthesis of AuNPs may be due to the edge-selective redu BPNS. Moreover, AuNPs can occupy the defects in BPNS to enhance the stability [38]. Raman spectroscopy could provide detailed vibrational and rotational pat sample identification. Raman spectra of BPNS and AuNPs-BPNS are provided i 3C. Three peaks are displayed at 363 cm −1 , 440 cm −1 and 466 cm −1 , which were asc one out-of-plane phonon mode Ag 1 and two in-plane phonon modes B2g and Ag respectively [39,40]. Upon AuNPs' modification, the three peaks blue-shifted because of vibration inhibition. Particularly, the B2g and Ag 2 mode shift was lar that of the Ag 1 mode, indicating that AuNPs-BPNS possess an in-plane heteros Furthermore, the changes in oxidation states of BPNS from pristine BPNS to BPNS were probed by XPS ( Figure S1). In Figure 3D, curves a and b display spectra before and after AuNP modification, respectively. The two unchanged pe 128.5 and 129.5 eV verify the original state of BP, which is consistent with stand The peak at 133 eV is due to oxidized phosphorus (i.e., POx) [41][42][43]. However, for BPNS (Figure 3Db), the POx peak was enhanced, demonstrating that some oxidized in the formation of AuNPs-BPNS. The experimental data coincide wi reported in the literature [38]. UV-Vis spectroscopy was conducted to characterize the formation process of the AuNPs. Figure 3A shows the UV-Vis spectrum of BPNS with different amounts of added HAuCl 4 . As the amount of BPNS increased, the absorption peaks of the AuNPs increased at 536 nm, demonstrating that the formation of the AuNPs is related to the BPNS. Additionally, the UV-Vis spectrum of HAuCl 4 added to BPNS with the increase in mixing time is shown in Figure 3B. The absorption peak intensity of the AuNPs increased with the increase in time, and the color of the solution changed to different shades of a red color. The successful synthesis of AuNPs may be due to the edge-selective reduction of BPNS. Moreover, AuNPs can occupy the defects in BPNS to enhance the stability of BPNS [38]. Raman spectroscopy could provide detailed vibrational and rotational patterns for sample identification. Raman spectra of BPNS and AuNPs-BPNS are provided in Figure 3C. Three peaks are displayed at 363 cm −1 , 440 cm −1 and 466 cm −1 , which were ascribed to one out-of-plane phonon mode A g 1 and two in-plane phonon modes B 2g and A g 2 for BP, respectively [39,40]. Upon AuNPs' modification, the three peaks blue-shifted slightly because of vibration inhibition. Particularly, the B 2g and A g 2 mode shift was larger than that of the A g 1 mode, indicating that AuNPs-BPNS possess an in-plane heterostructure. Furthermore, the changes in oxidation states of BPNS from pristine BPNS to AuNPs-BPNS were probed by XPS ( Figure S1). In Figure 3D, curves a and b display the P2p spectra before and after AuNP modification, respectively. The two unchanged peaks near 128.5 and 129.5 eV verify the original state of BP, which is consistent with standard BP. The peak at 133 eV is due to oxidized phosphorus (i.e., PO x ) [41][42][43]. However, for AuNPs-BPNS (Figure 3Db), the PO x peak was enhanced, demonstrating that some BP was oxidized in the formation of AuNPs-BPNS. The experimental data coincide with those reported in the literature [38].

Characterization of the Modified Electrode
EIS is an efficient method for exploring the interfacial properties of the m electrodes [44,45]. Therefore, the modification process was characterized by EIS to the electrodes' surface features. EIS measurement was performed in 5. K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) solution containing 0.1 M KCl. The equipment was mV of alternative voltage amplitude and 200 mV of applied potential, and the frequencies ranged from 0.01 Hz to 100 kHz. The electron transfer resistance (R expressed by the diameter of a semicircle. As shown in Figure 4A, the bare GCE s a small semicircle diameter (177 Ω, curve a), indicating a fast electron-transfer p When BPNS was immobilized on the GCE, the semicircle diameter became bigg that of bare GCE (704 Ω, curve b). As a result of the good electron transfer effici the AuNPs, the AuNPs-BPNS/GCE exhibited a lower Ret (566 Ω, curve c) than BPN suggesting that AuNPs could efficiently enhance the electron transfer efficiency. A assembly of the aptamer, the value of Ret increased to 1123 Ω (curve d) becau negatively charged aptamer and [Fe(CN)6] 3-/4-anions repelled each other. W hybridization between the aptamer and cDNA, the semicircle diameter further inc (curve e), which suggests the successful recognition of the aptamer and cDNA. The of EIS verified that the aptasensor was successfully prepared (Scheme 1). Furthe XPS spectra for the C, N, Au and S energy regions are shown in Figure S2, whi verified the aptamer immobilization on AuNPs-BPNS.
The SWV measurements were carried out to investigate the electrochemical be of cDNA/aptamer/AuNPs-BPNS/GCE in different conditions. As shown in Fig  only a low oxidation peak of MB at −0.25 V (curve a) can be observed in PBS w FMCA. After the addition of FMCA into PBS, a significant oxidation peak was pro

Characterization of the Modified Electrode
EIS is an efficient method for exploring the interfacial properties of the modified electrodes [44,45]. Therefore, the modification process was characterized by EIS to reveal the electrodes' surface features. EIS measurement was performed in 5.0 mM K 3 [Fe(CN) 6 ]/K 4 [Fe(CN) 6 ] (1:1) solution containing 0.1 M KCl. The equipment was set at 5 mV of alternative voltage amplitude and 200 mV of applied potential, and the voltage frequencies ranged from 0.01 Hz to 100 kHz. The electron transfer resistance (R et ) was expressed by the diameter of a semicircle. As shown in Figure 4A, the bare GCE showed a small semicircle diameter (177 Ω, curve a), indicating a fast electron-transfer process. When BPNS was immobilized on the GCE, the semicircle diameter became bigger than that of bare GCE (704 Ω, curve b). As a result of the good electron transfer efficiency of the AuNPs, the AuNPs-BPNS/GCE exhibited a lower R et (566 Ω, curve c) than BPNS/GCE, suggesting that AuNPs could efficiently enhance the electron transfer efficiency. After the assembly of the aptamer, the value of R et increased to 1123 Ω (curve d) because the negatively charged aptamer and [Fe(CN) 6 ] 3-/4anions repelled each other. With the hybridization between the aptamer and cDNA, the semicircle diameter further increased (curve e), which suggests the successful recognition of the aptamer and cDNA. The results of EIS verified that the aptasensor was successfully prepared (Scheme 1). Furthermore, XPS spectra for the C, N, Au and S energy regions are shown in Figure S2, which also verified the aptamer immobilization on AuNPs-BPNS. observed, demonstrating the hybridization among aptamer and cDNA. After PAT added to this mixture, the band of hybridization weakened but the band of cDNA obvious, which can be attributed to the initiation of hybridization and the releas cDNA. It implies that the aptamer and cDNA hybridized successfully and the apta could recognize PAT; thus, the cDNA was released.

Electrochemical PAT Assay
In order to obtain an excellent performance in sensing, some parameters w optimized: (a) the concentration of aptamer, (b) the hybridization time of aptamer cDNA, (c) the incubation time of PAT. Respective data and figures are given in electronic Supporting Material ( Figure S3). The following experimental conditions w found to give the best results: (a) optimal aptamer concentration: 1 μM; (b) opt hybridization time: 90 min; (c) optimal incubation time: 60 min.
The SWV was implemented to study the analytical performance of the propo aptasensor. As shown in Figure 5A, a series of different concentrations of PAT from nM to 100 μM were measured in PBS containing 10 μM FMCA. The peak current of increased and that of FMCA decreased with increasing PAT concentration. Figur displays the linear relationship between the individual MB or FMCA current resp and the logarithmic values of PAT concentration. For the MB signal, the limit of detec (LOD) was 0.011 ng/mL with R = 0.9987, and for the FMCA signal, 0.012 ng/mL with 0.9967. Furthermore, from Figure 5C  The SWV measurements were carried out to investigate the electrochemical behavior of cDNA/aptamer/AuNPs-BPNS/GCE in different conditions. As shown in Figure 4B, only a low oxidation peak of MB at −0.25 V (curve a) can be observed in PBS without FMCA. After the addition of FMCA into PBS, a significant oxidation peak was produced at 0.3 V (curve b). However, after incubation with PAT, the MB response increased significantly and the FMCA response decreased (curve c). The results reveal that the proposed aptasensor could be used for PAT detection. PAGE analysis was performed to demonstrate the DNA combination processes step by step. As shown in Figure S3 (Supporting Information), Lanes 1 and 2 correspond to the aptamer and cDNA, respectively. Lane 3 represents the mixture of aptamer and cDNA; a new band was observed, demonstrating the hybridization among aptamer and cDNA. After PAT was added to this mixture, the band of hybridization weakened but the band of cDNA was obvious, which can be attributed to the initiation of hybridization and the release of cDNA. It implies that the aptamer and cDNA hybridized successfully and the aptamer could recognize PAT; thus, the cDNA was released.

Electrochemical PAT Assay
In order to obtain an excellent performance in sensing, some parameters were optimized: (a) the concentration of aptamer, (b) the hybridization time of aptamer and cDNA, (c) the incubation time of PAT. Respective data and figures are given in the electronic Supporting Material ( Figure S3). The following experimental conditions were found to give the best results: (a) optimal aptamer concentration: 1 µM; (b) optimal hybridization time: 90 min; (c) optimal incubation time: 60 min.
The SWV was implemented to study the analytical performance of the proposed aptasensor. As shown in Figure 5A, a series of different concentrations of PAT from 0.1 nM to 100 µM were measured in PBS containing 10 µM FMCA. The peak current of MB increased and that of FMCA decreased with increasing PAT concentration. Figure 5B displays the linear relationship between the individual MB or FMCA current response and the logarithmic values of PAT concentration. For the MB signal, the limit of detection (LOD) was 0.011 ng/mL with R = 0.9987, and for the FMCA signal, 0.012 ng/mL with R = 0.9967. Furthermore, from Figure 5C, the linear relationship between the value of I MB /I FMCA and the logarithm of the PAT concentration was acquired as follows: I MB /I FMCA = 0.12 log C PAT + 0.38 with R = 0.9941. A good linear correlation was obtained from 0.0154 ng/mL to 15.4 µg/mL and the LOD reached 0.066 ng/mL (S/N = 3). Compared with either an MB or FMCA signal sensor, the dual-signal strategy has a lower LOD. (LOD) was 0.011 ng/mL with R = 0.9987, and for the FMCA signal, 0.012 ng/mL with R = 0.9967. Furthermore, from Figure 5C, the linear relationship between the value of IMB/IFMCA and the logarithm of the PAT concentration was acquired as follows: IMB/IFMCA = 0.12 log CPAT + 0.38 with R = 0.9941. A good linear correlation was obtained from 0.0154 ng/mL to 15.4 μg/mL and the LOD reached 0.066 ng/mL (S/N = 3). Compared with either an MB or FMCA signal sensor, the dual-signal strategy has a lower LOD. Moreover, the analytical performance for the proposed dual-signaling aptasensor is comparable with other methods in previous reports as can be seen in Table 1. This may be because BPNS has an excellent electronic performance and two-dimensional sheet structure with a large surface area which provides more sites for the generation of AuNPs and prevents AuNPs aggregation; in addition, the dual-signal ratio strategy greatly reduces the impact of the external environment on the detection results and improves the sensitivity of the sensor. The results indicate that the proposed aptasensor exhibits a lower LOD and wider linear range. The linearity in real spiked matrix samples was determined using pretreated fresh apple juice containing 10 µM FMCA and different PAT concentrations. The calibration curve ( Figure 6) was obtained from the linear regression of a plot of peak area versus concentration. Calibration was carried out in the range of 0.154-15,400 ng/mL, being found to be linear in the entire studied range with R = 0.9956.

Reproducibility, Stability and Selectivity
The reproducibility of the proposed aptasensor was evaluated by using five cDNA/ aptamer/AuNPs-BPNS/GCEs for the detection of 0.154 µg/mL PAT under the same conditions ( Figure 7A). The relative standard deviation (RSD) was calculated to be 4.5%, revealing significant reproducibility. Moreover, the aptasensor was stored under refrigeration for ten days. As shown in Figure 7B, 91.1% of the initial response of the current was obtained after adding the same amount of PAT (0.154 µg/mL), demonstrating the satisfactory stability of the proposed aptasensor.
The linearity in real spiked matrix samples was determined using pretreated fresh apple juice containing 10 μM FMCA and different PAT concentrations. The calibration curve ( Figure 6) was obtained from the linear regression of a plot of peak area versus concentration. Calibration was carried out in the range of 0.154-15,400 ng/mL, being found to be linear in the entire studied range with R = 0.9956.

Reproducibility, Stability and Selectivity
The reproducibility of the proposed aptasensor was evaluated by using five cDNA/aptamer/AuNPs-BPNS/GCEs for the detection of 0.154 μg/mL PAT under the same conditions ( Figure 7A). The relative standard deviation (RSD) was calculated to be 4.5%, revealing significant reproducibility. Moreover, the aptasensor was stored under  refrigeration for ten days. As shown in Figure 7B, 91.1% of the initial response of the current was obtained after adding the same amount of PAT (0.154 μg/mL), demonstrating the satisfactory stability of the proposed aptasensor. Furthermore, five kinds of contaminants in food samples containing hydroxymethylfurfural (HMF), aflatoxin B1 (AFB1), aflatoxin M1 (AFM), ochratoxin A (OTA) and zearalenone (ZEN) were used to verify the selectivity of the aptasensor. As shown in Figure 8, despite the fact that the concentrations of the interferences were as high as 10 μM, there were low values of IMB/IFMCA. However, an obvious response was obtained by the addition of PAT. The results indicated that the proposed dual-signaling aptasensor exhibits an excellent selectivity for PAT.  Furthermore, five kinds of contaminants in food samples containing hydroxymethylfurfural (HMF), aflatoxin B1 (AFB1), aflatoxin M1 (AFM), ochratoxin A (OTA) and zearalenone (ZEN) were used to verify the selectivity of the aptasensor. As shown in Figure 8, despite the fact that the concentrations of the interferences were as high as 10 µM, there were low values of I MB /I FMCA . However, an obvious response was obtained by the addition of PAT. The results indicated that the proposed dual-signaling aptasensor exhibits an excellent selectivity for PAT. refrigeration for ten days. As shown in Figure 7B, 91.1% of the initial response of the current was obtained after adding the same amount of PAT (0.154 μg/mL), demonstrating the satisfactory stability of the proposed aptasensor. Furthermore, five kinds of contaminants in food samples containing hydroxymethylfurfural (HMF), aflatoxin B1 (AFB1), aflatoxin M1 (AFM), ochratoxin A (OTA) and zearalenone (ZEN) were used to verify the selectivity of the aptasensor. As shown in Figure 8, despite the fact that the concentrations of the interferences were as high as 10 μM, there were low values of IMB/IFMCA. However, an obvious response was obtained by the addition of PAT. The results indicated that the proposed dual-signaling aptasensor exhibits an excellent selectivity for PAT.

Real Sample Detection
To evaluate the practical application capability of the present system, the proposed aptasensor was employed to detect the PAT contents in fresh and infected apple, pear,

Real Sample Detection
To evaluate the practical application capability of the present system, the proposed aptasensor was employed to detect the PAT contents in fresh and infected apple, pear, and tomato. As shown in Table 2, no PAT was found in the fresh foods, while small amounts were found in the infected foods. Then, the standard addition method was also performed. The standard PAT solutions were added into the food samples; the recoveries and RSDs were 95.0-106.7% and 2.9-5.1%. Furthermore, there was no significant difference between the results obtained by the aptasensor and an HPLC method, as shown in Table 1. These results implied that the dual-signaling aptasensor is applicable for PAT detection in food samples.

Conclusions
In summary, BPNS have potential value in the field of food safety detection. Metal nanoparticles, especially AuNPs, as an efficient catalyst, promote electron transfer on the AuNPs-BPNS, overcoming the defects of single metal nanoparticles. In this paper, in-plane gold nanoparticles-BPNS (AuNPs-BPNS) was synthesized, in which the high chemical activity of the defect sites in BPNS can edge-selective reduced HAuCl 4 , and the formed AuNPs not only enhance the stability of BPNS but also provide effective immobilization sites of the aptamer. The obtained AuNPs-BPNS nanocomposites were used to construct a dual-signaling aptasensor for PAT detection. Under optimum conditions, the proposed aptasensor was linear over the range of 0.0154 ng/mL-15.4 µg/mL. The electrochemical sensing platform has been successfully applied to the determination of PAT in apple, pear, and tomato extracts with good accuracy and reliability. Food safety detection methods based on functional nanomaterials will usher in new development opportunities in the future.